Abstract The rise of pathogens that are resistant to most antibiotics poses a major threat to public health and portends a return to the pre-antibiotic era. At present, there are several bacteria that can only be treated with a few antibiotics of last resort. One of the most widespread of these pathogens is methicillin resistant Staphylococcus aureus (MRSA), which is responsible for 80,000 invasive infections per year and an associated health care cost of $4 billion. MRSA has been treated primarily with vancomycin for over 50 years, yet surprisingly few cases of vancomycin resistant S. aureus (VRSA) have been reported. It remains unclear why VRSA has not spread between patients, although it is suspected that this is due to the slow growth rate of VRSA in the presence of vancomycin. Here we hypothesize that VRSA will overcome this fitness deficit through the acquisition of compensatory mutations during experimental evolution in a controlled laboratory setting. The goal of this project is to pinpoint these adaptive mutations, which may in the future enable us to take preemptive measures against the acquisition of resistance. Furthermore, we will investigate whether the observed compensatory mutations stabilize resistance, such that well-adapted VRSA is unlikely to revert to MRSA even if vancomycin was no longer used in the clinic. Hence, this project challenges the current clinical paradigm of confronting antibiotic resistance after it arises, potentially revealing a new tactic for maintaining the efficacy of antibiotics of last resort. If successful, we will identify factors that may predispose VRSA to spread, which could enable preemptive screening for these mutations and inform the development of control practices before an epidemic of resistance emerges. Moreover, this project would pioneer a new line of inquiry into the steps that must occur before reversion to sensitivity can be achieved in currently antibiotic-resistant pathogens. These outcomes are crucial for mitigating the risk that VRSA supplants MRSA as a dominant pathogen and, more generally, countering the rise of resistance to antibiotics of last resort. 2